56 Clinical and Laboratory Standards Institute. All rights reserved.

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1 Table 2C 56 Clinical and Laboratory Standards Institute. All rights reserved. Table 2C. Zone Diameter and Minimal Inhibitory Concentration Breakpoints for Testing Conditions Medium: Inoculum: diffusion: MHA Broth dilution: CAMHB; CAMHB + 2% NaCl for oxacillin; CAMHB supplemented to 50 µg/ml calcium for daptomycin Agar dilution: MHA; MHA + 2% NaCl for oxacillin. Agar dilution has not been validated for daptomycin. Direct colony suspension, equivalent to a 0.5 McFarland standard Incubation: 35 C 2 C; ambient air diffusion: 1618 hours; 24 hours (CoNS and cefoxitin) Dilution methods: 1620 hours; 24 hours for oxacillin and vancomycin; Testing at temperatures above 35 C may not detect MRS. General Routine QC Recommendations (See Tables 4A and 5A for acceptable QC ranges.) diffusion: Staphylococcus aureus ATCC Dilution methods: Staphylococcus aureus ATCC When a commercial test system is used for susceptibility testing, refer to the manufacturer s instructions for QC test recommendations and QC ranges. (1) For disk diffusion, test a maximum of 12 disks on a 150-mm plate and no more than 6 disks on a 100-mm plate; disks should be placed no less than 24 mm apart, center to center (see M02-A12, Subchapter 3.6). Each zone diameter should be clearly measurable; overlapping zones prevent accurate measurement. Measure the diameter of the zones of complete inhibition (as judged by the unaided eye), including the diameter of the disk. Hold the Petri plate a few inches above a black background illuminated with reflected light, except for linezolid, which should be read with transmitted light (plate held up to light source). The zone margin should be considered the area showing no obvious, visible growth that can be detected with the unaided eye. Ignore faint growth of tiny colonies that can be detected only with a magnifying lens at the edge of the zone of inhibited growth. With trimethoprim and the sulfonamides, antagonists in the medium may allow some slight growth; therefore, disregard slight growth (20% or less of the lawn of growth) and measure the more obvious margin to determine the zone diameter. For linezolid, any discernible growth within the zone of inhibition is indicative of resistance to the respective agent. (2) For staphylococci when testing chloramphenicol, clindamycin, erythromycin, linezolid, tedizolid, and tetracycline by broth microdilution MIC, trailing growth can make end-point determination difficult. In such cases, read the MIC at the lowest concentration where the trailing begins. Tiny buttons of growth should be ignored (see M07-A10, Figures 3 and 4). With trimethoprim and the sulfonamides, antagonists in the medium may allow some slight growth; therefore, read the end point at the concentration in which there is 80% reduction in growth as compared to the control (see M07-A10, Figure 2). (3) Historically, resistance to the penicillinase-stable penicillins (see Glossary I) has been referred to as methicillin resistance or oxacillin resistance. MRSAs are those strains of S. aureus that express meca or another mechanism of methicillin resistance, such as changes in affinity of penicillin-binding proteins for oxacillin (modified S. aureus strains). M100, 27th ed. For Use With M02-A12 and M07-A10

2 Clinical and Laboratory Standards Institute. All rights reserved. Table 2C. (Continued) (4) Most oxacillin resistance is mediated by meca, encoding the PBP 2a (also called PBP2'). Isolates that test positive for meca or PBP 2a should be reported as oxacillin resistant. S. aureus and CoNS isolates that test resistant by cefoxitin MIC or cefoxitin disk or demonstrate oxacillin MICs 4 g/ml should be reported as oxacillin resistant. For non-s. epidermidis CoNS with oxacillin MICs between 0.5 and 2 g/ml, see comment (15). Mechanisms of oxacillin resistance other than meca are rare and include a novel meca homologue, mecc. 1 S. epidermidis isolates with oxacillin MIC 0.5 g/ml should be reported as oxacillin resistant. MICs for strains with mecc are typically in the resistant range for cefoxitin and/or oxacillin; mecc resistance cannot be detected by tests directed at meca or PBP 2a. (5) Oxacillin-resistant S. aureus and CoNS (MRS), are considered resistant to other -lactam agents, ie, penicillins, -lactam/ -lactamase inhibitor combinations, cephems (with the exception of the cephalosporins with anti-mrsa activity), and carbapenems. This is because most cases of documented MRS infections have responded poorly to -lactam therapy, or because convincing clinical data that document clinical efficacy for those agents have not been presented. (6) Routine testing of urine isolates of S. saprophyticus is not advised, because infections respond to concentrations achieved in urine of antimicrobial agents commonly used to treat acute, uncomplicated UTIs (eg, nitrofurantoin, trimethoprim ± sulfamethoxazole, or a fluoroquinolone). (7) For tests for β-lactamase production, oxacillin resistance and meca-mediated oxacillin resistance using cefoxitin, reduced susceptibility to vancomycin, inducible clindamycin resistance, and high-level mupirocin resistance (S. aureus only), refer to Tables 3E, 3F, 3G, 3H and 3I, respectively. NOTE: Information in boldface type is new or modified since the previous edition. 1 García-Álvarez L, Holden MT, Lindsay H, et al. Methicillin-resistant Staphylococcus aureus with a novel meca homologue in human and bovine populations in the UK and Denmark: a descriptive study. Lancet Infect Dis. 2011;11(8): For Use With M02-A12 and M07-A10 M100, 27th ed. 57 Table 2C

3 Table 2C 58 Clinical and Laboratory Standards Institute. All rights reserved. Table 2C. (Continued) Content S I R S I R PENICILLINASE-LABILE PENICILLINS (8) Penicillin-susceptible staphylococci are susceptible to other β-lactam agents with established clinical efficacy for staphylococcal infections (including both penicillinase-labile and penicillinase-stable agents; see Glossary I). Penicillin-resistant staphylococci are resistant to penicillinase-labile penicillins. A Penicillin 10 units (9) Penicillin should be used to test the susceptibility of all staphylococci to all penicillinase-labile penicillins (see Glossary I). Penicillin-resistant strains of staphylococci produce -lactamase. Perform test(s) to detect -lactamase production on staphylococci for which the penicillin MICs are 0.12 µg/ml or zone diameters 29 mm before reporting the isolate as penicillin susceptible. Rare isolates of staphylococci that contain genes for -lactamase production may appear negative by -lactamase tests. Consequently, for serious infections requiring penicillin therapy, laboratories should perform MIC tests and -lactamase testing on all subsequent isolates from the same patient. PCR testing of the isolate for the blaz -lactamase gene may be considered. See Tables 3E and 3F. PENICILLINASE-STABLE PENICILLINS (10) For oxacillin-resistant staphylococci report penicillin as resistant or do not report. (11) Oxacillin (or cefoxitin) results can be applied to the other penicillinase-stable penicillins (cloxacillin, dicloxacillin, methicillin, and nafcillin). For agents with established clinical efficacy and considering site of infection and appropriate dosing, oxacillin (cefoxitin)-susceptible staphylococci can be considered susceptible to: -lactam/ -lactamase inhibitor combinations (amoxicillin-clavulanate, ampicillin-sulbactam, piperacillin-tazobactam) Oral cephems (cefaclor, cefdinir, cephalexin, cefpodoxime, cefprozil, cefuroxime, loracarbef) Parenteral cephems including cephalosporins I, II, III, and IV (cefamandole, cefazolin, cefepime, cefmetazole, cefonicid, cefoperazone, cefotaxime, cefotetan, ceftizoxime, ceftriaxone, cefuroxime, ceftaroline, moxalactam) Carbapenems (doripenem, ertapenem, imipenem, meropenem) Oxacillin-resistant staphylococci are resistant to all currently available -lactam antimicrobial agents, with the exception of the newer cephalosporins with anti-mrsa activity. Thus, susceptibility or resistance to a wide array of -lactam antimicrobial agents may be deduced from testing only penicillin and either cefoxitin or oxacillin. Testing of other -lactam agents, except those with anti-mrsa activity, is not advised. See general comments (4) and (5). Additional explanation on the use of cefoxitin for prediction of meca-mediated oxacillin resistance can be found in Subchapter 3.13 of M07-A10 and Subchapter 3.9 of M02-A12. M100, 27th ed. For Use With M02-A12 and M07-A10

4 Clinical and Laboratory Standards Institute. All rights reserved. Table 2C. (Continued) Content PENICILLINASE-STABLE PENICILLINS (Continued) A Oxacillin (For S. aureus and S. lugdunensis) 30 µg cefoxitin (surrogate test for oxacillin) S I R S I R (oxacillin) 4 (cefoxitin) 4 (oxacillin) 8 (cefoxitin) For use with S. aureus and S. lugdunensis. (12) Oxacillin disk testing is not reliable. See cefoxitin and general comment (5) for reporting oxacillin when testing cefoxitin as a surrogate agent. (13) Cefoxitin is tested as a surrogate for oxacillin; report oxacillin susceptible or resistant based on the cefoxitin result. (14) Cefoxitin MIC and disk diffusion tests performed on media other than CAMHB or unsupplemented MHA do not reliably detect meca-mediated resistance in isolates of S. aureus that do not grow on these media (eg, small colony variants). Testing for PBP2a using induced growth (ie, growth taken from the zone margin surrounding a cefoxitin disk on either BMHA or a blood agar plate after 24 hours incubation in 5% CO 2 ) or meca should be done. Isolates that test either meca negative or PBP2a negative or cefoxitin susceptible should be reported as oxacillin susceptible. For Use With M02-A12 and M07-A10 See general comment (4) and (5) and comments (8) and (11). M100, 27th ed. 59 Table 2C

5 Table 2C 60 Clinical and Laboratory Standards Institute. All rights reserved. Table 2C. (Continued) Content PENICILLINASE-STABLE PENICILLINS (Continued) A Oxacillin (For CoNS except S. lugdunensis and S. pseudintermedius) S I R S I R 0.25 (oxacillin) 30 µg cefoxitin (surrogate test for oxacillin) 0.5 (oxacillin) For use with CoNS except S. lugdunensis and S. pseudintermedius. (15) Oxacillin MIC breakpoints may overcall resistance for some CoNS, because some nons. epidermidis strains for which the oxacillin MICs are 0.52 µg/ml lack meca. For serious infections with CoNS other than S. epidermidis, testing for meca or for PBP 2a or with cefoxitin disk diffusion may be appropriate for strains for which the oxacillin MICs are µg/ml. Isolates that test either meca negative or PBP2a negative or cefoxitin susceptible should be reported as oxacillin susceptible See general comment (5) and comments (8), (11), and (13). A Oxacillin (For S. pseudintermedius) 1 g oxacillin (16) Neither cefoxitin MIC nor cefoxitin disk tests are reliable for detecting meca-mediated resistance in S. pseudintermedius. CEPHEMS (PARENTERAL) B Ceftaroline 30 µg (17) For reporting against S. aureus only, including MRSA. (18) Breakpoints are based on a dosage regimen of 600 mg every 12 h. M100, 27th ed. For Use With M02-A12 and M07-A10

6 Clinical and Laboratory Standards Institute. All rights reserved. Table 2C. (Continued) Content S I R S I R GLYCOPEPTIDES (19) For S. aureus, vancomycin-susceptible isolates may become vancomycin intermediate during the course of prolonged therapy. B B Vancomycin (For S. aureus) Vancomycin For use with S. aureus. (20) MIC tests should be performed to determine the susceptibility of all isolates of staphylococci to vancomycin. The disk test does not differentiate vancomycin-susceptible isolates of S. aureus from vancomycin-intermediate isolates, nor does the test differentiate among vancomycinsusceptible, -intermediate, and -resistant isolates of CoNS, all of which give similar size zones of inhibition. (21) Send any S. aureus for which the vancomycin is 8 g/ml to a reference laboratory. See Appendix A. Also refer to Table 3G for S. aureus, Subchapter in M07-A10, and Subchapter in M02-A For use with CoNS. For Use With M02-A12 and M07-A10 (For CoNS) See comment (20). (22) Send any CoNS for which the vancomycin MIC is 32 g/ml to a reference laboratory. See Appendix A. Inv. Teicoplanin See also Subchapter in M07-A10, and Subchapter in M02-A LIPOGLYCOPEPTIDES C Oritavancin 0.12 See comment (17). C Telavancin 0.12 See comment (17). LIPOPEPTIDES B Daptomycin 1 (23) Daptomycin should not be reported for isolates from the respiratory tract. M100, 27th ed. Table 2C

7 Table 2C 62 Clinical and Laboratory Standards Institute. All rights reserved. Table 2C. (Continued) Content S I R S I R AMINOGLYCOSIDES (24) For staphylococci that test susceptible, aminoglycosides are used only in combination with other active agents that test susceptible. C Gentamicin 10 g O Amikacin 30 g O Kanamycin 30 g O Netilmicin 30 g O Tobramycin 10 g MACROLIDES (25) Not routinely reported on organisms isolated from the urinary tract. A A A Azithromycin or clarithromycin or erythromycin 15 g 15 g 15 g O Telithromycin 15 g O Dirithromycin 15 g TETRACYCLINES (26) Organisms that are susceptible to tetracycline are also considered susceptible to doxycycline and minocycline. However, some organisms that are intermediate or resistant to tetracycline may be susceptible to doxycycline, minocycline, or both. B Tetracycline 30 g B Doxycycline 30 g B Minocycline 30 g See comment (25). FLUOROQUINOLONES (27) may develop resistance during prolonged therapy with quinolones. Therefore, isolates that are initially susceptible may become resistant within 3 to 4 days after initiation of therapy. Testing of repeat isolates may be warranted. C Ciprofloxacin or 5 g C levofloxacin 5 g C Moxifloxacin 5 g O Enoxacin 10 g (28) For testing and reporting of urinary tract isolates only. O Gatifloxacin 5 g O Grepafloxacin 5 g O Lomefloxacin 10 g O Norfloxacin 10 g See comment (28). O Ofloxacin 5 g O Sparfloxacin 5 g Inv. Fleroxacin 5 g NITROFURANTOINS U Nitrofurantoin 300 g M100, 27th ed. For Use With M02-A12 and M07-A10

8 Clinical and Laboratory Standards Institute. All rights reserved. Table 2C. (Continued) LINCOSAMIDES Content S I R S I R A Clindamycin 2 g (29) Inducible clindamycin resistance can be detected by disk diffusion using the D-zone test or by broth microdilution (see Table 3H, Subchapter in M02-A12, and Subchapter in M07-A10). See comment (25). FOLATE PATHWAY INHIBITORS A Trimethoprim- 1.25/23.75 g /38 4/76 sulfamethoxazole U Sulfonamides 250 or 300 g (30) Sulfisoxazole can be used to represent any of the currently available sulfonamide preparations. U Trimethoprim 5 g PHENICOLS C Chloramphenicol 30 g See comment (25). ANSAMYCINS B Rifampin 5 g (31) Rx: Rifampin should not be used alone for antimicrobial therapy. STREPTOGRAMINS O Quinupristin-dalfopristin 15 g (32) For reporting against methicillin-susceptible S. aureus. OXAZOLIDINONES B Linezolid 30 g (33) When testing linezolid, disk diffusion zones should be examined using transmitted light. Organisms with resistant results by disk diffusion should be confirmed using an MIC method. B Tedizolid See comment (17). Abbreviations: ATCC, American Type Culture Collection; BMHA, blood Mueller-Hinton agar; CAMHB, cation-adjusted Mueller-Hinton broth; CoNS, coagulasenegative staphylococci; I, intermediate; MHA, Mueller-Hinton agar; MIC, minimal inhibitory concentration; MRS, methicillin-resistant staphylococci; MRSA, methicillin-resistant S. aureus; PBP 2a, penicillin-binding protein 2a; PCR, polymerase chain reaction; QC, quality control; R, resistant; S, susceptible; UTI, urinary tract infection. For Use With M02-A12 and M07-A10 M100, 27th ed. 63 Table 2C